Light induced EPR spectra of reaction centers from Rhodobacter sphaeroides at 80K: Evidence for reduction of Q(B) by B-branch electron transfer in native reaction centers.

Photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides capture solar energy by electron transfer from primary donor, D, to quinone acceptor, Q(B,) through the active A-branch of electron acceptors, but not the inactive B-branch. The light induced EPR spectrum from native RCs that had Fe(2+) replaced by Zn(2+) was investigated at cryogenic temperature (80K, 35 GHz). In addition to the light induced signal due to formation of D(+•)Q(A) (-•) observed previously, a small fraction (~5%) of the signal displayed very different characteristics: (1) The signal was absent in RCs in which the Q(B) was displaced by the inhibitor stigmatellin. (2) Its decay time (τ=6 s) was the same as observed for D(+•)Q(B) (-•) in mutant RCs lacking Q(A,) which is significantly slower than for D(+•)Q(A) (-•) (τ=30 ms). (3) Its EPR spectrum was identical to that of D(+•)Q(B) (-•). (4) The quantum efficiency for forming the major component of the signal was the same as that found for mutant RCs lacking Q(A) (Φ =0.2%) and was temperature independent. These results are explained by direct photochemical reduction of Q(B)via B-branch electron transfer in a small fraction of native RCs.

Spectroscopic and kinetic properties of the transient intermediate acceptor in reaction centers of Rhodopseudomonas sphaeroides.

The photoreductive trapping of the transient, intermediate acceptor, I-, in purified reaction centers of Rhodopseudomonas sphaeroides R-26 was investigated for different external conditions. The optical spectrum of I- was found to be similar to that reported for other systems by Shuvalov and Klimov ((1976) Biochim. Biophys. Acta 400, 587--599) and Tiede et al. (P.M. Tiede, R.C. Prince, G.H. Reed and P.L. Dutton (1976) FEBS Lett. 65, 301--304). The optical changes of I- showed characteristics of both bacteriopheophytin (e.g. bleaching at 762, 542 nm and red shift at 400 nm) and bacteriochlorophyll (bleaching at 802 and 590 nm). Two types of EPR signals of I- were observed: one was a narrow singlet at g = 2.0035, deltaH = 13.5 G, the other a doublet with a splitting of 60 G centered around g = 2.00, which was only seen after short illumination times in reaction centers reconstituted with menaquinone. The optical and EPR kinetics of I- on illumination in the presence of reduced cytochrome c and dithionite strongly support the following three-step scheme in which the doublet EPR signal is due to the unstable state DI-Q-Fe2+ and the singlet EPR signal is due to DI-Q2-Fe2+. : formula: (see text), where D is the primary donor (BChl)2+. The above model was supported by the following observations: (1) During the first illumination, sigmoidal kinetics of the formation of I- was observed. This is a direct consequence of the three-sequential reactions. (2) During the second and subsequent illuminations first-order (exponential) kinetics were observed for the formation of I-. This is due to the dark decay, k4, to the state DIQ2-Fe2+ formed after the first illumination. (3) Removal of the quinone resulted in first-order kinetics. In this case, only the first step, k1, is operative. (4) The observation of the doublet signal in reaction centers containing menaquinone but not ubiquinone is explained by the longer lifetime of the doublet species I-(Q-Fe2%) in reaction centers containing menaquinone. The value of tau2 was determined from kinetic measurements to be 0.01 s for ubiquinone and 4 s for menaquinone (T = 20 degrees C). The temperature and pH dependence of the dark electron transfer reaction I-(Q-Fe2+) yields I(Q2-Fe2+) was studied in detail. The activation energy for this process was found to be 0.42 eV for reaction centers containing ubiquinone and 0.67 eV for reaction centers with menaquinone. The activation energy and the doublet splitting were used to calculate the rate of electron transfer from I- to MQ-Fe2+ using Hopfield's theory for thermally activated electron tunneling. The calculated rate agrees well with the experimentally determined rate which provides support for electron tunneling as the mechanism for electron transfer in this reaction. Using the EPR doublet splitting and the activation energy for electron transfer, the tunneling matrix element was calculated to be 10(-3) eV. From this value the distance between I- and MQ- was estimated to be 7.5--10 A.

ENDOR studies of the intermediate electron acceptor radical anion I-. in Photosystem II reaction centers.

The EPR and ENDOR characteristics of the intermediate electron acceptor radical anion I-. in Photosystem II (PS II) are shown to be identical in membrane particles and in the D1D2 cytochrome b-559 complex (Nanba, O. and Satoh, K. (1987) Proc. Natl. Acad. Sci. USA 84, 109-112). These findings provide further evidence that the D1D2 complex is the reaction center of PS II and show that the pheophytin binding site is intact. A hydrogen bond between I-. and the protein (GLU D1-130) is postulated on the basis of D2O exchange experiments. The ENDOR data of I-. and of the pheophytin a radical anion in different organic solvents are compared and the observed differences are related to structural changes of the molecule on the basis of molecular orbital calculations (RHF-INDO/SP). The importance of the orientation of the vinyl group (attached to ring I) on electron transfer is discussed.

Electron nuclear double resonance of semiquinones in reaction centers of Rhodopseudomonas sphaeroides.

Replacement of Fe2+ by Zn2+ in reaction centers of Rhodopseudomonas sphaeroides enabled us to perform ENDOR (electron nuclear double resonance) experiments on the anion radicals of the primary and secondary ubiquinone acceptors (QA- and QB-. Differences between the QA and QB sites, hydrogen bonding to the oxygens, interactions with the protons of the proteins and some symmetry properties of the binding sites were deduced from an analysis of the ENDOR spectra.

EPR study of the electronic properties and weak exchange interactions in bis(L-phenylalaninamidato)Cu(II).

We performed EPR measurements in powder and single crystal samples of bis(L-phenylalaninamidato)Cu(II) (C18H22CuN4O2). We evaluated the crystal and molecular g-factors, and estimate an exchange interaction of 0.15 K< or = J(AB)/k < or =0.32 K between coppers spaced apart 6.24 A, transmitted through an amidate bridge Cu-O-C-N-Cu. An unusual line shape is observed in powder samples arising from the relative orientation of symmetry-related molecules in the structure. The dipolar interaction and the layered structure of the copper ions produce a strong temperature variation of the spectral shape when the copper spins are polarized by the applied magnetic field.

Primary acceptor in bacterial photosynthesis: obligatory role of ubiquinone in photoactive reaction centers of Rhodopseudomonas spheroides.

Reaction centers were found to bind two ubiquinones, both of which could be removed by o-phenanthroline and the detergent lauryldimethylamine oxide. One ubiquinone was more easily removed than the other. The low-temperature light-induced optical and electron paramagnetic resonance (EPR) changes were eliminated and restored upon removal and readdition of ubiquinone and were quantitatively correlated with the amount of tightly bound ubiquinone. We, therefore, conclude that this ubiquinone plays an obligatory role in the primary photochemistry. The easily removed ubiquinone is thought to be the secondary electron acceptor. The low-temperature charge recombination kinetics, as well as the optical and EPR spectra, were the same for untreated reaction centers and for those reconstituted with ubiquinone. This indicates that extraction and reconstitution were accomplished without altering the conformation of the active site. Reaction centers reconstituted with other quinones also showed restored photochemical activity, although they exhibited changes in their low-temperature recombination kinetics and light-induced (g = 1.8) EPR signal is interpreted in terms of a magnetically coupled ubiquinone--Fe2+ acceptor complex. A possible role of iron is to facilitate electron transfer between the primary and secondary ubiquinones.

Electron paramagnetic resonance investigation of photosynthetic reaction centers from Rhodobacter sphaeroides R-26 in which Fe2+ was replaced by Cu2+. Determination of hyperfine interactions and exchange and dipole-dipole interactions between Cu2+ and QA-.

We report electron paramagnetic resonance (EPR) experiments in frozen solutions of unreduced and reduced photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides R-26 in which Fe2+ has been chemically replaced by the isotope 65Cu2+. Samples in which the primary quinone acceptor QA is unreduced (Cu2+QA:RCs) give a powder EPR spectrum typical for Cu2+ having axial symmetry, corresponding to a d(x2 - y2) ground state orbital, with g values g parallel = 2.314 +/- 0.001 and g perpendicular = 2.060 +/- 0.003. The spectrum shows a hyperfine structure for the nuclear spin of copper (65I = 3/2) with A parallel = (-167 +/- 1) x 10(-4) cm-1 and /A perpendicular/ = (16 +/- 2) x 10(-4) cm-1, and hyperfine couplings with three nitrogen ligands. This has been verified in samples containing the naturally occurring 14N isotope (l = 1), and in samples where the nitrogen ligands to copper were replaced by the isotope 15N (l = 1/2). We introduce a model for the electronic structure at the position of the metal ion which reflects the recently determined three-dimensional structure of the RCs of Rb. sphaeroides (Allen, J. P., G. Feher, T. O. Yeates, H. Komiya, and D. C. Rees. 1987. Proc. Natl. Acad. Sci. USA. 84:5730: Allen, J. P., G. Feher, T. O. Yeates, H. Komiya, and D. C. Rees. 1988. Proc. Natl. Acad. Sci. USA, 85:8487) as well as our EPR results. In this model the copper ion is octahedrally coordinated to three nitrogens from histidine residues and to one carboxylate oxygen from a glutamic acid, forming a distorted square in the plane of the d(x2 = y2) ground state orbital. It is also bound to a nitrogen of another histidine and to the other carboxylate oxygen of the same glutamic acid residue, in a direction approximately normal to this plane. The EPR spectrum changes drastically when the quinone acceptor QA is chemically reduced (Cu2+QA-:RCs); the change is due to the exchange and dipole-dipole interactions between the Cu2+ and QA- spins. A model spin Hamiltonian proposed for this exchange coupled cooper-quinone spin dimer accounts well for the observed spectra. From a comparison of the EPR spectra of the Cu2+QA:RC and CU2+QA-:RC complexes we obtain the values /J0/ = (0.30 +/- 0.02) K for the isotropic exchange coupling, and /d/ = (0.010 +/- 0.002) K for the projection of the dipole-dipole interaction tensor on the symmetry axis of the copper spin. From the EPR experiments only the relative signs of J0 and d can be deduced; it was determined that they have the same sign. The magnitude of the exchange coupling calculated for Cu2+QA-:RC is similar to that observed for the Fe2+QA-:RC complex (J0 = -0.43K). The exchange coupling is discussed in terms of the superexchange paths connecting the Cu2+ ion and the quinone radical using the structural data for the RCs of Rb. sphaeroides. From the value of the dipole-dipole interaction, d, we determined R approximately 8.4 A for the weighted distance between the metal ion and the quinone in reduced RCs, which is to be compared with 10 A obtained from x-ray analysis of unreduced RCs. This points to a shortening of the Cu2+ -QA- distance upon reduction of the quinone, as has been proposed by Allen et al. (1988).

Reaction of nitric oxide with heme proteins: studies on metmyoglobin, opossum methemoglobin, and microperoxidase.

Kinetic and EPR studies show that the first step in the reaction of NO with ferric myoglobin, opossum hemoglobin, and microperoxidase is the reversible formation of the H-NO complex: H + NO in equilibrium H-NO (where H = Mb+, or Hb+ OP, or MP+). The NO-combination rates are markedly affected by the presence or absence of the distal histidine. The distal histidine significantly reduces the NO-combination rates, perhaps by interaction between the distal histidine and the ferric iron. Thus the beta-chains of Hb+ OP and metmyoglobin show similar combination rates. In the absence of a distal histidine, the NO-combination rates in the alpha-chains of Hb+ OP are much faster and similar to those observed for the five-coordinate heme in microperoxidase. The loss of a water molecule from the six-coordination site is assumed to be the rate-limiting step.

15N electron nuclear double resonance of the primary donor cation radical P+.865 in reaction centers of Rhodopseudomonas sphaeroides: additional evidence for the dimer model.

Four 15N hyperfine coupling constants, including signs, have been measured by electron nuclear double resonance (ENDOR) and electron nuclear nuclear triple resonance (TRIPLE) for the bacteriochlorophyll a radical cation, BChla+., in vitro and for the light-induced primary donor radical cation, P+.865, in reaction centers of Rhodopseudomonas sphaeroides R-26. A comparison of the data shows that the hyperfine coupling constants have the same sign in both radicals and are, on the average, smaller by a factor of 2 in P+.865. These results provide additional evidence that P+.865 is a bacteriochlorophyll dimer and are in contradiction with the monomer structure of P+.865 recently proposed by O'Malley and Babcock. The reduction factors of the individual 15N couplings, together with the evidence from proton ENDOR data and molecular orbital calculations, indicate a dimer structure in which only two rings (either I and I or III and III) of the bacteriochlorophyll macrocycles overlap.

Electronic structure of Q-A in reaction centers from Rhodobacter sphaeroides. I. Electron paramagnetic resonance in single crystals.

The magnitude and orientation of the electronic g-tensor of the primary electron acceptor quinone radical anion, Q-A, has been determined in single crystals of zinc-substituted reaction centers of Rhodobacter sphaeroides R-26 at 275 K and at 80 K. To obtain high spectral resolution, EPR experiments were performed at 35 GHz and the native ubiquinone-10 (UQ10) in the reaction center was replaced by fully deuterated UQ10. The principal values and the direction cosines of the g-tensor axes with respect to the crystal axes a, b, c were determined. Freezing of the single crystals resulted in only minor changes in magnitude and orientation of the g-tensor. The orientation of Q-A as determined by the g-tensor axes deviates only by a few degrees (< or = 8 degrees) from the orientation of the neutral QA obtained from an average of four different x-ray structures of Rb. sphaeroides reaction centers. This deviation lies within the accuracy of the x-ray structure determinations. The g-tensor values measured in single crystals agree well with those in frozen solutions. Variations in g-values between Q-A, Q-B, and UQ10 radical ion in frozen solutions were observed and attributed to different environments.

The electronic structure of Fe2+ in reaction centers from Rhodopseudomonas sphaeroides. III. EPR measurements of the reduced acceptor complex.

Electron paramagnetic resonance (EPR) spectra of the reduced quinone-iron acceptor complex in reaction centers were measured in a variety of environments and compared with spectra calculated from a theoretical model. Spectra were obtained at microwave frequencies of 1, 9, and 35 GHz and at temperatures from 1.4 to 30 K. The spectra are characterized by a broad absorption peak centered at g = 1.8 with wings extending from g approximately equal to 5 to g less than 0.8. The peak is split with the low-field component increasing in amplitude with temperature. The theoretical model is based on a spin Hamiltonian, in which the reduced quinone, Q-, interacts magnetically with Fe2+. In this model the ground manifold of the interacting Q-Fe2+ system has two lowest doublets that are separated by approximately 3 K. Both perturbation analyses and exact numerical calculations were used to show how the observed spectrum arises from these two doublets. The following spin Hamiltonian parameters optimized the agreement between simulated and observed spectra: the electronic g tensor gFe, x = 2.16, gFe, y = 2.27, gFez = 2.04, the crystal field parameters D = 7.60 K and E/D = 0.25, and the antiferromagnetic magnetic interaction tensor, Jx = -0.13 K, Jy = -0.58 K, Jz = -0.58 K. The model accounts well for the g value (1.8) of the broad peak, the observed splitting of the peak, the high and low g value wings, and the observed temperature dependence of the shape of the spectra. The structural implications of the value of the magnetic interaction, J, and the influence of the environment on the spin Hamiltonian parameters are discussed. The similarity of spectra and relaxation times observed from the primary and secondary acceptor complexes Q-AFe2+ and Fe2+Q-B leads to the conclusion that the Fe2+ is approximately equidistant from QA and QB.

Co-crystallization and characterization of the photosynthetic reaction center-cytochrome c2 complex from Rhodobacter sphaeroides.

The photosynthetic reaction center (RC) of Rhodobacter sphaeroides and cytochrome c2 (cyt c2), its physiological secondary electron donor, have been co-crystallized. The molar ratio of RC/cyt c2 was found by SDS-PAGE and optical absorbance changes in the co-crystals to be 4. The crystals diffracted X-rays to 3.5 angstroms. However, the resolution degraded during data collection. A data set, 82.5% complete, was collected to 4.5 angstroms. The crystals belong to the tetragonal space group P4(3)2(1)2, with unit cell dimensions of a = b = 142.7 angstroms and c = 254.8 angstroms. The positions of the RCs in the unit cell were determined by molecular replacement. A comparable search for the cyt c2 by this method was unsuccessful because of the small contribution of the cytochrome to the total scattering and because of its low occupancy. The cyt c2 was positioned manually into patches of difference electron density, adjacent to the periplasmic surface of the M polypeptide subunit of the RC. The difference electron density was not sufficient for precise positioning of the cyt c2, and its orientation was modeled by placing the exposed edge of the heme toward the primary donor of the reaction center D and by forming pairs for electrostatically interacting RC and cyt c2 amino acid residues. The RC-cyt c2 structure derived from the co-crystal data was supported by use of omit maps and structure refinement analyses. Cyt c2 reduces the photooxidized primary donor D+ in 0.9 +/- 0.1 micros in the co-crystals, which is the same as the fast electron transfer rate in vivo and in solution. This result provides strong evidence that the structure of the complex in the co-crystal is the same as in solution. Two additional methods were used to investigate the structure of the RC-cyt c2 complex: (i) Docking calculations based on interprotein electrostatic interactions identified possible binding positions of the cyt c2 on the RC. The cyt c2 position with the lowest electrostatic energy is very similar to that of the cyt c2 in the proposed co-crystal structure. (ii) Site-directed mutagenesis was used to modify two aspartic acid residues (M184 and L155) on the periplasmic surface of the RC. Cyt c2 binding affinity to these RCs and electron transfer rates to D+ in these RCs support the co-crystal structure of th RC-cyt c2 complex.